Aminoacyl transfer RNA synthetases arose early in evolution and established the rules of the genetic code through their specific aminoacylations of transfer RNAs. the enzymes are organized into two classes of ten enzymes each. These two classes appear to have no historical relationship to each other. Experiments are proposed to investigate and manipulate the tRNA specificity of class I and class II enzymes. These synthetases include E. coli alanine, glutamine, isoleucine and methionine tRNA synthetases, and S. cerevisiae glutamine tRNA synthetase. Recent experiments have shown that the amino acid acceptor helix is a major determinant of the identity of at least some tRNAs. Functional synthetase-tRNA acceptor helix contacts will be identified and manipulated through analysis of mutant enzymes which are selected for specific alterations in their acceptor helix interactions. A minimalist aminoacyl tRNA synthetase will also be designed and subjected to mutagenesis and selection for functional activity. Additional efforts will be directed at manipulations of tRNA recognition specificity by introducing the fewest possible amino acid or nucleotide substitutions, in some cases taking advantage of recent x-ray crystallographic structural information.